Project Summary Mammalian cells package two meters of linear DNA into a nucleus whose diameter is on average only a few microns. This packaging must be done in such a way that DNA transactions such as transcription, replication, and repair can faithfully occur. The development of the `Hi-C' technique has enabled the mapping of chromosome-chromosome interaction frequency on the genome-wide scale in large populations of cells. Hi- C has generated detailed datasets that have revealed key organizational features, including `domains' of elevated interaction frequency and the broad partitioning of the genome into active and inactive `compartments.' Disruption of these features is associated with cancer and developmental disease. However, in part due to technical limitations, pressing open questions remain about the causes and consequences of 3D genome organization, particularly in single cells. For instance, it is not fully clear whether features such as domains actually form in individual cells, or instead represent the averaging of many individual states that are present in the population of cells profiled because single-cell Hi-C methods provide very sparse datasets in which most features of genome organization remain invisible. Furthermore, the physical properties of these genomic features have yet to be examined in single cells, and it is not currently known if these regions create distinct diffusive environments that can impact the activity of trans-acting factors. Finally, the discovery of additional proteins that influence nuclear architecture has been limited by the high financial and resource cost of Hi-C. Our previous work established a powerful new platform for investigating 3D genome organization in single cells using microscopy. We introduced a programmable fluorescent in situ hybridization (FISH) approach that uses sets of bioinformatically designed oligonucleotide probes to create complex hybridization patterns in fixed samples. We have harnessed this technology to pioneer the use of single-molecule super- resolution microscopy to study chromosome structure on the nanoscale, introduced a technique capable of visually distinguishing homologous chromosomes, and also have developed an approach that facilitates the multiplexed amplification of fluorescent signals in fixed cells and tissues. We will build upon this foundational work by developing and applying single-cell imaging technologies and leveraging advanced optical approaches to directly address these unsolved questions. Specifically, we will introduce a broadly enabling single-cell imaging strategy capable of resolving features of 3D genome organization with unprecedented detail using multiplexed FISH (Objective 1) and investigate the biophysical properties of 3D organizational features with live-cell imaging (Objective 2). We will augment these efforts with high-throughput, unbiased screening approaches to identify new regulators of 3D genome structure (Objective 3). Our detailed characterization of nuclear architecture and its biophysical consequences will also provide an enhanced framework for understanding how alterations in 3D genome organization can lead to human disease.